This invention relates to an improved process for detecting a specific isozyme of lactate dehydrogenase, termed LDH.sub.k in the serum of cancer patients. More particularly, this invention relates to an improved assay for quantitatively determining the level of LDH.sub.k in human serum, to aid in diagnosing the presence of cancer and to monitor the success of cancer therapy.
The enzyme lactate dehydrogenase (LDH; L-lactate : NAD+oxidoreductase) catalyzes the reaction: EQU Pyruvate+NADH.sub.2 .revreaction.Lactate+NAD
Lactate dehydrogenase has a number of isozymes; isozymes are enzymes possessing the same or a similar catalytic function, but are distinguishable by either biochemical, physical or immunological means. The first isozymes of lactate dehydrogenase were found to be tetramers composed of two distinct polypeptide parent chains termed H (heart) and M (muscle), named for the organs from which they are readily obtained. The H and M subunits combine to give rise to the five classical isozymes of LDH. The five tetrameric isozymes of LDH are: H.sub.4, H.sub.3 M, H.sub.2 M.sub.2, and M.sub.4. At pH 7, H.sub.4 is the most negatively charged and thus appears nearest the anode after zone electrophoresis, while M.sub.4 appears nearest the cathode. H.sub.4 functions predominantly in the aerobic oxidation of lactate and is found in high concentration in heart muscle, erythrocytes, and the renal cortex. M.sub.4 is primarily involved in the reduction of pyruvate and anaerobic metabolism, and is found in highest concentrations in skeletal muscle, liver, skin and the ileum.
Another isozyme, termed X.sub.4, has been isolated from spermatozoa and testis, and is a tetramer composed of another subunit form of LDH, referred to as the X subunit. This subunit is encoded by a third gene separate from those encoding the synthesis of the H and M subunits. This, along with the fact that the other five isozymes have been electrophoretically resolved into numerous subbands, suggests that the molecular nature of lactate dehydrogenase is complex.
In 1981, another isozyme of LDH was characterized in cells transformed by the Kirsten murine sarcoma virus (KiMSV). This unique isozyme was designated LDH.sub.k. It has been isolated in anaerobically stressed rat cells and in Kirsten sarcoma virus-infected non-rat cells. LDH.sub.k purified from cells transformed by a temperature-sensitive transforming gene mutant of KiMSV was thermolabile relative to LDH.sub.k purified from cells infected by wild type virus. Therefore, it is thought to actually be encoded by KiMSV, and most likely by its transforming gene. [Anderson, G. R. et al., J. Biol. Chem. 256: 10583-10591 (1981)].
LDH.sub.k is readily distinguishable from the other known isozymes of LDH. Specifically, it is essentially inactive if assayed under a normal atmosphere due to inhibition by oxygen, has subunits of 35,000 and 22,000 daltons (which appear to cleave from a 57,000--dalton precursor), is more basic than other LDH isozymes and is inhibited by guanosine triphosphate (GTP) and more strongly by the related compounds 5', 5' diadenosine tetraphosphate (AP.sub.4 A) and 5', 5' diguanosine tetraphosphate (Gp4G).
The discovery that human tumors contain high levels of LDH.sub.k activity was first reported in 1981 [Anderson, G. R. et al., Proc. Nat. Acad. Sci. USA 78: 3209-3213, 1981]. In this study, 16 different human carcinomas were analyzed for the presence of LDH.sub.k, and 11 out out of the 16 tumor tissues tested showed a 10 to 500-fold increase over the LDH.sub.k activity seen in adjoining nontumor tissues. In contrast, other LDH isozymes exhibited only a 2 to 5-fold increase in the tumor tissues as compared with nontumor tissue. These researchers analyzed tumor tissues from human colon, mammary, laryngeal, renal, parotid and stomach carcinomas, and malignant melanoma. The LDH.sub.k in these human tumor tissues was inactive in the presence of oxygen, being activated by the presence of nitrogen or in the presence of cyanide, and was inhibited by GTP. These results suggested that LDH.sub.k might be clinically useful as a diagnostic tumor marker, particularly if it were released outside the tumor tissue.
At the present time there are several biochemical substances known to be secreted by tumor cells and released outside the tumor, which are sufficiently different in quantity or quality from products of normal cells to act as "tumor markers". One such marker is carcinoembryonic antigen (CEA) which is utilized to detect adenocarcinomas of the digestive system, particularly carcinoma of the colon. A very sensitive radioimmunoassay has been developed to detect small amounts of CEA in the blood which assay procedure has proven to be valuable in the early diagnosis of colon carcinoma and in detecting its recurrence after surgery.
A clinical spectrophotometric assay for LDH activity is presently utilized for monitoring myocardial, liver and hematologic disorders. Numerous studies have been done to investigate alterations in serum levels of the classical isozymes of LDH in human cancer, and assays of the classical isozymes of LDH in human tumor tissues have also been done. A definite and consistent shift in the pattern of molecular forms of LDH has been found in a series of malignant human neoplasms as compared with benign tumors and normal controls. Although evidence has been found for an increase in total LDH activity in malignant tumors, metastatic nodules have been shown to have lower LDH activity levels and different LDH compositions when compared with their associated primary tumors. Accordingly, due to the irregular patterns of expression of the classical LDH isozymes in human cancer serum, assay for these isozymes is not presently used as an aid in detecting or monitoring the progression of malignant diseases. It should be noted that since LDH.sub.k is inactive when assayed in the presence of oxygen, it would not have been detected in any of the previous studies.
Preliminary unpublished data showed that LDH.sub.k is released into the serum of cancer patients. Therefore, it was thought that a quantitative assay procedure for the expression of LDH activity in human serum might be useful in the clinical realm. However, conventional clinical assaying techniques have not been sufficiently sensitive to permit a meaningful quantitative determination of LDH.sub.k activity in human serum.